Luciferases can generate light via the oxidation of enzyme-specific substrates, e.g., luciferins. This provides the basis for assays directed at detecting the presence or level of luciferin in a sample. In one configuration, the luciferin detected is produced by a non-luciferase enzyme of interest converting a pro-luciferin substrate to a luciferin product. For firefly luciferase and all other beetle luciferases, light generation occurs in the presence of luciferin, magnesium ions, oxygen, and ATP. For anthozoan luciferases, including Renilla luciferase, only oxygen is required along with the substrate coelenterazine. Resultant bioluminescence, if any, is then measured using a luminometer or any suitable radiant energy-measuring device.
Beetle luciferase substrates, e.g., luciferin, contain a carboxylic acid group that is necessary for the efficient production of light. However, a variety of enzymes do not utilize substrates having a carboxylic acid group near the reaction site (e.g. CYP2D6) making assay of these enzymes very difficult using substrates containing a carboxylic acid group. For in vitro assays/reactions, this problem can be overcome by esterifying the carboxylic acid group, thus neutralizing its charge, on the substrate. However, this increases the size of the substrate, and thus may prevent its entry to the active site of the enzyme of interest resulting in a compound that is an ineffective substrate for the enzyme of interest.
Even if the esterification of a substrate does produce a substrate which can be used effectively by an enzyme of interest in an in vitro assay, such a substrate may not work in cell-based assays, e.g., live cells, as esterases present in the cell rapidly cleave esters thereby releasing the carboxylic acid form of the substrate which, as mentioned above, may not be an acceptable form of substrate for the enzyme of interest.
In the present invention, a new form of substrate, i.e., a derivative of 2-cyano-6-substituted benzothiazole, is disclosed for use in cell-based assays for the detection or measurement of an enzyme(s) of interest, i.e. non-luciferase enzyme(s). The substrates in the present invention do not contain the carboxylic acid group normally found on known luciferin substrates, but instead are precursors of luciferin analogs that can be rapidly and quantitatively transformed to luciferins by addition of D-Cysteine. In this way, the substrates of the present invention are smaller in size than those of the corresponding luciferin analogs yet do not have the negatively charged group found in know luciferins. The present invention also provides methods of using the substrates of the present invention in cell-based assays to detect or measure non-luciferase enzymes of interest.